Ebook Histotechnology - A self-Instructional text (3rd edition): Part 1

Histotechnology
A Self-Instructional Text
3rd Edition


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Histotechnology
A Self-Instructional Text
3rd Edition

Freida L Carson
PhD, HT(ASCP)
Department of Pathology (retired)
Baylor University Medical Center
Dallas, Texas

Christa Hladik
AA, HT(ASCP)'m, QIHC
Clinical Laboratory Manager,
Neuropathology and Immunohistochemistry,
UT Southwestern Clinical Laboratories,
University of Texas Southwestern Medical Center
Dallas, Texas

•

American Society for
Clinical Pathology

Press


Publishing Team
Adam Fanucci (Illustrations)
Erik N Tanck & Tae W Moon (Design/Production)
Joshua Weikersheimer (Publishing direction)

Notice
Trade names for equipment and supplies described are included as suggestions only. In no way does their inclusion constitute an
endorsement of preference by the Author or the ASCP. The Author and ASCP urge all readers to read and follow all manufacturers'
instructions and package insert warnings concerning the proper and safe use of products. The American Society for Clinical Pathology,
having exercised appropriate and reasonable effort to research material current as of publication date, does not assume any liability for
any loss or damage caused by errors and omissions in this publication. Readers must assume responsibility for complete and thorough
research of any hazardous conditions they encounter, as this publication is not intended to be all-inclusive, and recommendations and
regulations change over time.

Cover Images
Image (left) : Hematoxylin eosin (H&E) - small intestine
Image (middle): Papanicolaou- cervical smear
Image (right): Aldan yellow-toluidine blue- gastric biopsy showing H pylori

•

American Society for
Clinical Pathology
Press
Copyright© 2009 by the American Society for Clinical Pathology. All rights reserved. No part of this publication may be reproduced,
stored in a retrieval system, or transmitted in any form or by any means electronic, mechanical, photocopying, recording, or otherwise,
without the prior written permission of the publisher.

Printed in Hong Kong
13 12 11 10 09

iv


Table of Contents
xvi

Preface

............................................
Chapter I
............................................
Fixation

16

POTASSIUM DICHROMATE (K 2Cr20 7 )

16

ZINC SALTS (ZnS0 4)

17

Other Fixative Ingredients

17

17
17
17
19

2

Definition

19

20

2

Functions of Fixatives

20

2

Actions of Fixatives

4

Factors Affecting Fixation

20
20
20

4

TEMPERATURE

4

SIZE

4

VOLUME RATIO

5

TIME

21
21

21
21

7

CHOICE OF FIXATIVE

21

7

PENETRATION

22

7

TISSUE STORAGE

7

pH

7

OSMOLALITY

8

Reactions of the Cell with Fixatives

8

THE NUCLEUS

8

PROTEIN

8

LIPIDS

9

CARBOHYDRATES

9

Simple Aqueous Fixatives or Fixative
Ingredients

9

ACETIC ACID

9
10
10

12
12
12
10
10

12
12

FORMALDEHYDE

12% Aqueous Formalin
12% Formalin Saline
Calcium Formalin
Formalin Ammonium Bromide
Acetate Formalin
12% Neutralized Formalin
12% Neutral-Buffered Formalin
Modified Millonig Formalin
Alcoholic Formalin

13

GLUTARALDEHYDE

13
14

Phosphate-Buffered Glutaraldehyde
GLYOXAL (C 2 H 20 2)

14

MERCURIC CHLORIDE (HgCl 2)

15

OSMIUM TETROXIDE (OsO 4 )

15

PICRIC ACID

Compound or Combined Fixatives
B-5 FIXATIVE
Stock Solution
Working Solution
Bouin Solution
Gendre Solution
Hollande Solution
ZENKER AND HELLY (ZENKER-FORMOL)
SOLUTIONS
Zenker and Helly Stock Solution
Zenker Working Solution
Helly Working Solution
Orth Solution
Zamboni Solution (Buffered Picric Acid-Formaldehyde,
or PAF)
ZINC FORMALIN SOLUTIONS
Aqueous Zinc Formalin (original formula)
Unbuffered Aqueous Zinc Formalin
Alcoholic Zinc Chloride Formalin

22

Nonaqueous Fixatives

22

ACETONE

22

ALCOHOL

22

23
23
23
23
23
23
25

Carnoy Solution
Clarke Fluid

Transport Solutions
Michel Transport Medium
PBS Buffer Stock Solution (also used in
immunohistochemistry)
PBS-10% Sucrose Solution

Removal of Fixation Pigments
Lugo) Iodine Solution

24

Troubleshooting Fixation Problems

24

AUTOLYSIS

24

INCOMPLETE FIXATION

27

References

Histotechnology 3rd Edition v


............................................
Chapter 2
............................................
Processing

Special Techniques in Processing

46
46

DECALCIFICATION

49

TROUBLESHOOTING DECALCIFICATION

49

FROZEN SECTIONS

50

TROUBLESHOOTING PROCESSING TISSUE FOR

33

Dehydration

33

ALCOHOLS

34

ACETONE

34

UNIVERSAL SOLVENTS

35
35

Clearing

35

TOLUENE

35

BENZENE

36

CHLOROFORM

54

Microscopes

36

ACETONE

54

LIGHT MICROSCOPE

36

ESSENTIAL OILS

55

POLARIZING MICROSCOPE

36

LIMONENE REAGENTS (XYLENE SUBSTITUTE)

55

PHASE-CONTRAST MICROSCOPE

36

ALIPHATIC HYDROCARBONS (XYLENE

55

DARKFIELD MICROSCOPE

56

FLUORESCENCE MICROSCOPE

56

ELECTRON MICROSCOPE

XYLENE

SUBSTITUTE)

FROZEN SECTIONS

51

References

............................................
Chapter 3
................
................... .........
Instrumentation

36

UNIVERSAL SOLVENTS

37

OTHER CLEARING AGENTS

57

Microtomes

37

Infiltration

57

ROTARY MICROTOME

37

PARAFFIN

57

SLIDING MICROTOME

40

WATER-SOLUBLE WAXES

57

CLINICAL FREEZING MICROTOME

40

CELLOIDIN

58

MICROTOME BLADES

41

PLASTICS

58

TROUBLESHOOTING MICROTOMY

41

AGAR AND GELATIN

30

41% SUCROSE

64

Cryostat

41
41

Troubleshooting Processing

65
65

Tissue Processors

66

MICROWAVE PROCESSOR

41

OVERDEHYDRATION

42

POOR PROCESSING

42

SPONGE ARTIFACT

67
67

AUTOMATIC STAINER

42

TISSUE ACCIDENTALLY DESICCATED

67

MICROWAVE STAINING OVEN

68

AUTOMATIC COVERSLIPPER

PRECIPITATE IN THE PROCESSOR CHAMBER

AND IN THE TUBING

CONVENTIONAL PROCESSOR

Stainers and Coverslippers

42

Embedding and Specimen Orientation

44

69
69

Miscellaneous Equipment

Troubleshooting Embedding

44

SOFT MUSHY TISSUE

70

CHROMIUM POTASSIUM SULFATE-COATED

45

INCORRECT ORIENTATION

46

TISSUE CARRYOVER

70

POLY-L-LYSINE-COATED SLIDES

46

TISSUE NOT EMBEDDED AT THE SAME LEVEL

70

AMINOALKYLSILANE-TREATED SLIDES

46

PIECES OF TISSUE MISSING FROM THE BLOCK

71

DRYERS AND OVENS

72

CIRCULATING WATER BATH

72

FREEZERS AND REFRIGERATORS

72

pH METERS

74

BALANCES AND SCALES

74

EMBEDDING CENTER

vi

FLOTATION BATHS
SLIDES


75

MICROMETER PIPETTES

75

SOLVENT RECYCLER

75

75

Instrument Quality Control

75

QUALITY CONTROL PROGRAM

16

............................................
~~~~t:r. s. .................................... .

Laboratory Mathematics
and Solution Preparation

NEW INSTRUMENT VALIDATION
94

Percentage Solutions

95

Use of the Gravimetric Factor in
Solution Preparation

96

Hydrates

96

Normal and Molar Solutions

References

............................................
~~~J:!t~r. ~ .................................... .

Safety
82

Biological or Infectious Hazards

82

TUBERCULOSIS EXPOSURE

82

CRYOGENIC SPRAYS

83

HIV, HEPATITIS C VIRUS (HCV), AND HBV

83

CREUTZFELDT-JAKOB DISEASE (CJD)

83

HANDLING TISSUE WASTE

83

Mechanical Hazards

84

ERGONOMICS

84

Chemical Hazards

86

PARTICULARLY HAZARDOUS SUBSTANCES
(REPRODUCTIVE TOXINS, SELECT
CARCINOGENS, AND SUBSTANCES WITH A
HIGH DEGREE OF ACUTE TOXICITY)

86

CARCINOGENS

86

CORROSIVE SUBSTANCES

86

FIRE AND EXPLOSION HAZARDS

87

HAZARDOUS CHEMICAL SPILLS AND STORAGE

88

CHEMICAL STORAGE

88

HAZARDOUS CHEMICAL DISPOSAL

89

Hazard Identification
General Safety Practices

90
90

EMPLOYEES

90

SUPERVISORS

91

References

97

The Metric System

97

TEMPERATURE CONVERSION

98

Buffers

98

General Guidelines for Solution
Preparation, Use, and Storage

99

Stability of Solutions

99

References

101

ANSWERS TO PROBLEMS IN CHAPTER

101

ANSWERS TO PROBLEMS IN LEARNING
ACTIVITIES

............................................
~~~~t:r. ~ .................................... .

Nuclear and Cytoplasmic
Staining
104

Ultrastructure of the Cell

104

THE NUCLEUS

105

THE CYTOPLASM

101

Staining Mechanisms

107

NUCLEAR STAINING

107

CYTOPLASMIC STAINING

108

The Dyes

109

FACTORS AFFECTING DYE BINDING

109

DIFFERENTIATION

109

THE NUCLEAR DYES

110

Harris Hematoxylin
Delafield Hematoxylin
Mayer Hematoxylin
Ehrlich Hematoxylin

111
111

111

Histotechnology 3rd Edition vii


112
112
113
113

Gill Hematoxylin
Scott Solution
Weigert Hematoxylin
Celestine Blue

114

PLASMA STAINS
Eosin Counterstain
Eosin-Phloxine B Counterstain

114

H&E Staining

114

MANUAL PROGRESSIVE STAINING METHOD

115

MANUAL REGRESSIVE STAINING METHOD

116

AUTOMATED STAINING

113
113

130

'troubleshooting Mounted Stained
Sections

130

WATER BUBBLES NOTED IN MOUNTED
SECTIONS

130

ALL AREAS OF SECTION CANNOT BE BROUGHT
INTO FOCUS

130

CORN-FLAKING ARTIFACT SEEN ON MOUNTED

131

MOUNTED STAINED SECTIONS ARE NOT

SECTIONS
AS CRISP AS USUAL WHEN VIEWED
MICROSCOPICALLY
RETRACTED MOUNTING MEDIUM

References

11 7

FROZEN SECTION STAINING

131

118

Troubleshooting the H&E Stain

132

118

INCOMPLETE DEPARAFFINIZATION

118

NUCLEAR STAINING IS NOT CRISP

11 9

PALE NUCLEAR STAINING

119

DARK NUCLEAR STAINING

120

RED OR RED-BROWN NUCLEI

120

PALE CYTOPLASMIC STAINING

136

Carbohydrates

120

DARK CYTOPLASMIC STAINING

136

GROUP 1: NEUTRAL POLYSACCHARIDES

120

EOSIN NOT PROPERLY DIFFERENTIATED

121

BLUE-BLACK PRECIPITATE ON TOP OF SECTIONS

121

HAZY OR MILKY WATER AND SLIDES

122

UNEVEN H&E STAINING

122

DARK BASOPHILIC STAINING OF NUCLEI AND
CYTOPLASM, ESPECIALLY AROUND TISSUE

............................................
Chapter 7
...............
.... .. ............ .. ...... ...
Carbohydrates and Amyloid
(NONIONIC HOMOGLYCANS)
136

GROUP II: ACID MUCOPOLYSACCHARIDES
(ANIONIC HETEROGLYCANS)

136

GROUP III: GLYCOPROTEINS (MUCINS, MUCOID,
MUCOPROTEIN, MUCOSUBSTANCES)

136

GROUP IV: GLYCOLIPIDS

137

Special Staining Techniques

EDGES
122

POOR CONTRAST BETWEEN NUCLEUS AND
CYTOPLASM

137
137

123
123
123
123

123

Nucleic Acid Stains
FEULGEN REACTION
Hydrochloric Acid, IN
Schiff Reagent (De Tomasi Preparation)
Sulfurous Acid

125

METHYL GREEN-PYRONIN Y
Solution a-0.2M Acetic Acid
Methyl Green-Pyronin Y Staining Solution

126

Polychromatic Stains

124
125

138
139
140
140
140
141
141
142
142

127
127
127
127
127
127

MAY-GRUNWALD GIEMSA STAIN
Stock Jenner Solution
Working Jenner Solution
Stock Giemsa Solution
Working Giemsa Solution
Acetic Water, 1%

143
143
143
145
145
146
146

128

Mounting Stained Sections

128

RESINOUS MEDIA

129

AQUEOUS MOUNTING MEDIA

129

COVER SLIPS

viii

146
146
147
147
147

PAS REACTION
Periodic Acid, 0.5% Solution
Schiff Reagent
PAS REACTION WITH DIASTASE DIGESTION
Periodic Acid, 0.5% Solution
Potassium Metabisulfite, 0.55% Solution
Phosphate Buffer, pH 6
BEST CARMINE
Carmine Stock Solution
Working Carmine Solution
MAYER MUCICARMINE
Mucicarmine Stock Solution
Mucicarmine Working Solution

Weigert Iron Hematoxylin
ALCIAN BLU E, PH 2.5
Acetic Acid, 3% Solution
ALCIAN BLUE, PH 1.0
O.lN Hydrochloric Acid Solution
1% Alcian Blue Solution, pH 1.0
Nuclear-Fast Red Solution
ALCIAN BLUE WITH H YALURONIDASE
O.lM Potassium Phosphate, Monobasic
Nuclear-Fast Red Solution


148
148
149
149
149
150
150
150

ALCIAN BLUE-PAS-HEMATOXYLIN

152
152
153
154
154
155
155

Amyloid

157

Acetic Acid, 3% Solution
Aldan Blue, pH 2.5
Schiff Reagent
MULLER-MOWRY COLLOIDAL IRON
Ferric Chloride, 29% Solution
Working Colloidal Iron Solution
Nuclear-Fast Red Solution

172
173
173
173
174
174
175
175
176
177

ALKALINE CONGO RED METHOD
Stock 80% Alcohol Saturated with Sodium Chloride
CRYSTAL VIOLET
Stock Saturated Crystal Violet Solution

Iodine-Iodide Solution

Crocein Scarlet-Acid Fuchsin Solution
Phosphotungstic Acid, 5% Solution
Alcoholic Safran Solution
SILVER TECHNIQUES FOR RETICULAR FIBERS
GOMORI STAIN FOR RETICULAR FIBERS
Silver Nitrate, 10% Solution
Potassium Permanganate, 0.5% Solution
Sodium Thiosulfate, 2% Solution
GORDON AND SWEETS STAIN FOR RETICULAR
FIBERS

178
178
178

Silver Nitrate, 10% Solution
Potassium Permanganate, 1% Solution
Ferric Ammonium Sulfate, 2.5% Solution

179
179

MALLORY PTAH TECHNIQUE FOR

THIOFLAVINE T FLUORESCENT METHOD
Thioflavine T, 1% Solution

References

............................................

Chapter 8
............................................
Connective
and Muscle Tissue

Staining Techniques for Muscle
CROSS-STRIATIONS AND FIBRIN

180
180
180
180
181
181

PTAH Solution
Gram Iodine
Sodium Thiosulfate, 5% Solution
Potassium Permanganate, 0.25% Solution
PTAH WITHOUT MERCURIC SOLUTIONS
Acidic Dichromate Solution

160

Connective 'tissue

182

Staining Technique for Basement
Membranes

161

Basement Membrane

182

PERIODIC ACID-METHENAMINE SILVER
MICROWAVE PROCEDURE FOR BASEMENT

161

Muscle

162

~taining

162
163
164
165
165
166
166
167
168
168
168
168

170
170
171
171
171

MASSON TRICHROME STAIN

172
172
172

Techniques for Connective
Tissue Fibers

Bouin Solution
Light Green Counterstain
GOMORI 1-STEP TRICHROME STAIN
Bouin Solution
Acetic Acid, 0.5% Solution
VAN GIESON PICRIC ACID-ACID FUCHSIN STAIN
Acid Fuchsin, 1% Solution

MEMBRANES

182
182
183
183
184

184
185
185
186
186

VERHOEFF ELASTIC STAIN

187

Lugo) Iodine
Ferric Chloride, 10% Solution
Sodium Thiosulfate, 5% Solution

187

ALDEHYDE FUCHSIN ELASTIC STAIN
Aldehyde Fuchsin Solution
Alcoholic Basic Fuchsin, 0.5% Solution
Aldehyde Fuchsin Solution
NOTES ON OTHER ELASTIC STAINS
RUSSELL MODIFICATION OF THE MOVAT
PENTACHROME STAIN
Aldan Blue, 1% Solution
Alkaline Alcohol Solution

Stock Methenamine Silver
Gold Chloride, 0.02% Solution
Methenamine Silver Solution
Gold Chloride, 0.2% solution

Staining Techniques for Lipid
OIL RED 0 METHOD FOR NEUTRAL FATS
Oil Red 0 Stock Solution
Oil Red 0 Working Solution
SUDAN BLACK B IN PROPYLENE GLYCOL
Calcium-Formalin Solution
OSMIUM TETROXIDE PARAFFIN PROCEDURE
FOR FAT
Osmium Tetroxide, 1% Solution

188

Staining Techniques for Connective
'tissue Cells

188
188
188

TOLUIDINE BLUE FOR MAST CELLS

190

References

Toluidine Blue Solution
METHYL GREEN-PYRONIN Y

Histotechnology 3rd Edition ix

Chapter 9
............................................
Nerve

202

NERVE FIBERS, NEUROFIBRILLARY TANGLES,
AND SENILE PLAQUES: MICROWAVE
MODIFICATION OF BIELSCHOWSKY METHOD

203
203

194

The Nervous System

194

Neurons

194

NISSL SUBSTANCE

204

194

NERVE CELL PROCESSES

205

194

Neuroglia

204

Silver Nitrate, 1% Solution
Sodium Thiosulfate, 2% Solution
NERVE FIBERS, NEUROFIBRILLARY TANGLES,
AND SENILE PLAQUES: THE SEVIER-MUNGER
MODIFICATION OF BIELSCHOWSKY METHOD

205

194

OLIGODENDROGLIA

194

ASTROCYTES

207

195

MICROGLIA
EPENDYMAL CELLS

Myelin

195

Special Staining Techniques

195

NISSL SUBSTANCE: CRESYL ECHT VIOLET
METHOD I

195
195
196

Cresyl Echt Violet Solution
Balsam-Xylene Mixture
NISSL SUBSTANCE: CRESYL ECHT VIOLET
METHOD II

196
196
197

Stock Cresyl Echt Violet Solution
Working Cresyl Echt Violet Solution, pH 2.5

197
197
198
199

Protargol, 1% Solution
Oxalic Acid, 2% Solution
Aniline Blue Solution
NERVE FIBERS AND NEUROFIBRILS: HOLMES
SILVER NITRATE METHOD

199
200
200

Aqueous Silver Nitrate, 20% Solution
Reducing Solution
NERVE FIBERS, NEUROFIBRILLARY TANGLES,
AND SENILE PLAQUES: BIELSCHOWSKY-PAS

GLIAL FIBERS: MALLORY PHOSPHOTUNGSTIC
ACID HEMATOXYLIN (PTAH) STAIN

208
208

GLIAL FIBERS: HOLZER METHOD

208

209
210
210

Aqueous Phosphomolybdic Acid, 0.5% Solution
ASTROCYTES: CAJAL STAIN

Formalin Ammonium Bromide

211

MYELIN SHEATH: WEIL METHOD

211

Ferric Ammonium Sulfate, 4% Solution

212

213
214

MYELIN SHEATH: LUXOL FAST BLUE METHOD

Luxol Fast Blue, 0.1% Solution
MYELIN SHEATH AND NISSL SUBSTANCE
COMBINED: LUXOL FAST BLUE-CRESYL ECHT

NERVE FIBERS, NERVE ENDINGS,

NEUROFIBRILS: BODIAN METHOD

Potassium Permanganate, 0.25% Solution

PTAH Solution
Lugol Iodine
Potassium Permanganate, 1% Solution
Oxalic Acid, 5% Solution

207
2 07

195

NEUROFIBRILLARY TANGLES AND SENILE
PLAQUES: THIOFLAVIN S (MODIFIED)

2 06

195

Silver Nitrate, 20% Solution
Sodium Thiosulfate, 5% Solution

214
215

VIOLET STAIN
Acetic Acid, 10% Solution
MYELIN SHEATHS AND NERVE FIBERS

COMBINED: LUXOL FAST BLUE-HOLMES
SILVER NITRATE METHOD

216
216
216
218
218
218

Aqueous Silver Nitrate, 20% Solution
Impregnating Solution
Lithium Carbonate, 0.05% Solution
LUXOL FAST BLUE-PAS-HEMATOXYLIN

Luxol fast blue, 0.1% Solution
Periodic Acid, 0.5% Solution

STAIN
201
201
201
2 01
201
201
20 I

x

Aqueous Silver Nitrate, 20% Solution

Ammoniacal Silver Solution
Developer
Gold Chloride, 0.5% Solution
Sodium Thiosulfate, 5% Solution
Periodic Acid, 1% Solution
Schiff Reagent

219

References


Chapter 10
............................................
Microorganisms

222

Bacteria

222

Fungi

223

Viruses

239

GROCOTT METHENAMINE-SILVER NITRATE
FUNGUS STAIN

240
240
240
242

Chromic Acid, 5% Solution
Silver Nitrate, 5% Solution
Sodium Thiosulfate, 2% Solution
MICROWAVE METHENAMINE-SILVER NITRATE
PROCEDURE FOR FUNGI

242
244

Chromic Acid, 10% Solution
MAYER MUCICARMINE AND ALCIAN
BLUE TECHNIQUES E FOR CRYPTOCOCCUS
NEOFORMANS

224

Protozoans

244

224
224

224
226

Special Staining Techniques

244
245
245

WARTHIN-STARRY TECHNIQUE FOR
SPIROCHETES

KINYOUN ACID-FAST STAIN
Kinyoun Carbol-Fuchsin Solution

226
227

Ziehl-Neelsen Carbol-Fuchsin Solution
MICROWAVE ZIEHL-NEELSEN METHOD FOR
ACID-FAST BACTERIA

227
228

ORGANISMS

228
229
230

246
246
246
247

Xylene-Peanut Oil
Ziehl-Neelsen Carbol-Fuchsin Solution

247
248
248
249

Auramine 0-Rhodamine B Solution
Acid Alcohol, 0.5% Solution
BROWN-HOPPS MODIFICATION OF THE GRAM
STAIN

231
231
233

GIEMSA METHODS

233

MODIFIED DIFF-QUIK GIEMSA STAIN FOR

Crystal Violet, 1% Solution

Gram Iodine

HELICOBACTER PYLORI

233
233
233
234

Diff-Quik Solution I
Diff-Quik Solution II
Acetic Acid Water
ALCIAN YELLOW-TOLUIDINE BLUE METHOD
FOR H PYLORI

234
235
235
236
236
237
237
238
238
238

Periodic acid, 1% Solution
HOTCHKISS-MCMANUS PAS REACTION FOR
FUNGI
Periodic Acid, 1% Solution

IN Hydrochloric Acid
CHROMIC ACID-SCHIFF STAIN FOR FUNGI (CAS)
Chromic acid, 5% Solution
Fast Green, 1:5000 Solution

DIETERLE METHOD FOR SPIROCHETES AND
Alcoholic Uranyl Nitrate, 5% Solution
Alcoholic Gum Mastic, 10% Solution
Formic Acid, 10% Solution
MICROWAVE STEINER AND STEINER
PROCEDURE FOR SPIROCHETES,

MICROWAVE AURAMINE-RHODAMINE

HELICOBACTER, AND LEGIONELLA ORGANISMS

FLUORESCENCE TECHNIQUE

230
230
231

Glycine-Acetic Acid Stock Solution
Silver Nitrate, 2% Solution
Hydroquinone, 0.1% Solution
LEGIONELLA ORGANISMS

Carbol-Fuchsin Solution
FITE ACID-FAST STAIN FOR LEPROSY

MICROWAVE MODIFICATION OF THE
WARTHIN-STARRY METHOD FOR BACTERIA

ZIEHL-NEELSEN METHOD FOR ACID-FAST
BACTERIA

Citric Acid, 1% Solution
Gelatin, 5% Solution

249
251

Uranyl Nitrate, 1% Solution

References

............................................
Chapter 11
............................................
Pigments, Minerals,
and Cytoplasmic Granules
254
254

Pigments

254

EXOGENOUS PIGMENTS

254

ENDOGENOUS HEMATOGENOUS PIGMENTS

255

ENDOGENOUS NONHEMATOGENOUS PIGMENT

255

Endogenous Deposits

256

Minerals

256

Cytoplasmic Granules

256
256
257
257

Special Staining Techniques

ARTIFACT PIGMENT S

GRIDLEY FUNGUS STAIN

Chromic Acid, 4% Solution
Aldehyde Fuchsin Solution

PRUSSIAN BLUE STAIN FOR FERRIC IRON
Potassium Ferrocyanide, 2% Solution
Nuclear-Fast Red (Kernechtrot) Solution
Histotechnology 3rd Edition xi


258

TURNBULL BLUE STAIN FOR FERROUS IRON

258

Hydrochloric Acid, 0.06N Solution
SCHMORL TECHNIQUE FOR REDUCING

259

SUBSTANCES
259
259
260

Ferric Chloride, 1% Stock Solution
Metanil Yellow, 0.25% Solution
FONTANA-MASSON STAIN FOR MELANIN AND
ARGENTAFFIN GRANULES

261
261
261
262
262
262
263
264
264
265

Silver Nitrate, 10% Solution
Gold Chloride, 0.2% Solution
MICROWAVE FONTANA-MASSON STAIN

Fontana Silver Nitrate Solution
Gold Chloride, 0.2% Solution
Sodium Thiosulfate, 2% Solution
GRIMELIUS ARGYROPHIL STAIN

Silver Nitrate, 1% Solution
Nuclear-Fast Red Solution
CHURUKIAN-SCHENK METHOD FOR
ARGYROPHIL GRANULES

265

Citric Acid, 0.3% Solution

266

MICROWAVE CHURUKIAN-SCHENK METHOD

266

Citric Acid-Glycine Stock Solution
GOMORI METHENAMINE-SILVER METHOD FOR

Chapter 12
............................................
Immunohistochemistry
278

Introduction

278

General Immunology

278

ANTIBODY

278

ANTIGEN

278

POLYCLONAL ANTISERA

278

MONOCLONAL ANTIBODIES

279

RABBIT MONOCLONAL ANTIBODIES

219

Tissue Handling

279

FROZEN TISSUE FIXATION AND PROCESSING

280

FIXATIVES FOR PARAFFIN-PROCESSED TISSUE

280

PROCESSING

280

MICROTOMY

281

EPITOPE ENHANCEMENT OR RETRIEVAL

283

Methods of Visualization

283

IMMUNOFLUORESCENCE

283

ENZYME IMMUNOHISTOCHEMISTRY

FOR ARGYROPHIL GRANULES
267

URATES
267
268
268
269

269
270
270
271
271
272

272
273
273

214

Silver Nitrate, 5% Solution
Stock Methenamine-Silver Nitrate Solution

284

BILE STAIN

Ferric Chloride, 10% Solution
VON KOSSA CALCIUM STAIN

Silver Nitrate, 5% Solution
ALIZARIN RED S CALCIUM STAIN

284

DIRECT METHOD

284

INDIRECT METHOD

284

UNLABELED, OR SOLUBLE ENZYME IMMUNE

COMPLEX, METHOD

Alizarin Red S Stain, 2% Solution
RHODANINE METHOD FOR COPPER

Saturated Rhodanine Solution (Stock)

Immunohistochemical Staining
Methods

284

AVIDIN-BIOTIN METHODS

285

POLYMERIC DETECTION

MICROWAVE RHODANINE COPPER METHOD

Saturated Rhodanine Solution (Stock)
Sodium Borate (Borax), 0.5%

285

Controls

285

POSITIVE CONTROLS

285

NEGATIVE CONTROLS

285

Antibody Evaluation and Validation

285

ANTIBODY SPECIFICATION SHEET

285

PREDILUTED AND CONCENTRATED

References

ANTIBODIES
286

ANTIBODY VALIDATION

287

STORAGE OF ANTIBODIES

287

BLOCKING REACTIONS

289

MULTILINK BIOTINYLATED SECONDARY
ANTISERA

xii

289

DAB REACTION PRODUCT INTENSIFICATION

289

BUFFER SOLUTIONS


289

Commonly Used Antibodies and Their
Applications

289

NEOPLASTIC TERMINOLOGY

289

Quality Control

290

RECOMMENDED QC FOR AN ANTIBODY

291

POSITIVE AND NEGATIVE TISSUE CONTROLS

292

RECOMMENDED QC FOR A TISSUE BLOCK

292

DAILY QC OF IMMUNOHISTOCHEMISTRY

292

STORAGE OF CONTROL SLIDES

292

Standardization

296

'troubleshooting Immunoperoxidase
Techniques

291
297
297
298
298
299
300
300
300

308

Muscle Histology

308

Pathologic Changes in Muscle

309

Enzyme Histochemistry

310

Oxidation and Reduction

310

Properties of Enzymes

310

Preservation of Enzymes

Staining Techniques

310

Classification of Enzymes

BASIC PAP IMMUNOPEROXIDASE PROCEDURE

311

HYDRO LASES

312

OXIDOREDUCTASES

312

TRANSFERASES

312

Freezing Muscle Biopsy Specimens

314

a-NAPHTHYL ACETATE ESTERASE STAIN FOR

Modified PBS Buffer (Stock Solution)
Primary Antibodies
Swine Antirabbit Linking Serum
ABC-IMMUNOPEROXIDASE PROCEDURE

Modified PBS Buffer (Stock Solution)
AEC
Acetate Buffer (O.OSM, pH 5.2)

301

HRP ENZYME-LABELED POLYMER PROCEDURE

301

Tris-Buffered Saline Solution (with Tween-TBST), pH 7.6
Ready to Use
Primary Antibodies
Chromogen Solution
Retrieval Solution (pH 6.0)

302
302
302
303

Chapter 13
............................................

Enzyme Histochemistry

References

315
315
315
315
315
315
315
316

MUSCLE BIOPSIES
0.2N Phosphate Buffer, pH 7.2
Pararosaniline Stock Solution
Sodium Nitrite, 4% Solution
a-Naphthyl Acetate in Acetone, 1% Solution
lNHCl
IN Sodium Hydroxide
Incubation Solution (prepare just before use)
NAPHTHOL AS-D CHLOROACETATE ESTERASE
TECHNIQUE

316
317
317
317
317
317

Esterase Solution A
Esterase Solution B
Esterase Solution C
O.lN HCl
O.IM Sodium Barbital (Sodium Diethylbarbiturate)
Working Esterase Solution

317

MAYER HEMATOXYLIN

318

ATPASE STAIN

318
318
318
318
319
320
321
321
322
322
323

Barbital Acetate Buffer Stock Solution A
Barbital Acetate Buffer Stock Solution B (O.lN HCl)

Barbital Acetate Buffer Working Solution
Sodium Barbital Solution (use to make 10.4, 9.4, and
incubation solutions)
Incubating Solution
ACID PHOSPHATASE IN MUSCLE BIOPSIES

2N HCl
Incubating Medium
ALKALINE PHOSPHATASE STAIN FOR MUSCLE
BIOPSIES

0.2M Tris
Incubating Medium

Histot.echnology 3rd Edition xiii


323
323
324
325
325
326
326
326

NADH DIAPHORASE
Saline Solution
Phosphate Buffer, pH 7.4
SUCCINIC DEHYDROGENASE (SDH)

Phosphate Buffer, 0.2M, pH 7.6
Physiological Saline
PHOSPHORYLASE STAIN FOR MUSCLE

Nonenzymatic Procedures for Muscle
Disorders

328
328

MODIFIED GOMORI TRICHROME
Gomori Trichrome Solution

329

Acknowledgment

330

References

............................................
Chapter 14
............................................
Electron Microscopy
334
334

Fixation

335

FACTORS INFLUENCING FIXATION

335
335
335

FIXATIVE SOLUTIONS

336
336
336
336

Paraformaldehyde with Cacodylate Buffer
Paraformaldehyde or Glutaraldehyde with Phosphate
Buffer
Formaldehyde with Phosphate Buffer (Modified Millonig
Fixative)
Formaldehyde-Glutaraldehyde (4CF-1G)
Buffered PAF (Zamboni Fixative)
Osmium Tetroxide with Cacodylate Buffer
Osmium Tetroxide with Phosphate Buffer

Processing

337

TRANSITIONAL SOLVENTS

337

EMBEDDING MEDIA

337

PROCEDURE FOR ROUTINE PROCESSING AND

DEHYDRATION

SPURR EMBEDDING
PROCEDURE FOR ROUTINE PROCESSING AND
EPON EMBEDDING

339

PROCEDURE FOR LR WHITE PROCESSING FOR
ELECTRON MICROSCOPY IMMUNOLABELING

340
341

Sectioning

341

KNIVES

342

CORRECTING PROBLEMS ENCOUNTERED IN
SECTIONING

xiv

343
343
343
344
344

Staining 0.5-µm Sections

345
345

Staining Thin Sections

346
346

Special Techniques

347

CELL SUSPENSIONS (FLUIDS, CULTURES,

TOLUIDINE BLUE-BASIC FUCHSIN PROCEDURE
Staining Solution

TOLUIDINE BLUE STAINING
Toluidine Blue, 2%

Lead Citrate Solution

BLOOD CELL PREPARATION
PARASITES, ETC)

347

Processing Tissues Previously
Embedded in Paraffin

347

Processing Tissue from an
H&E-Stained Paraffin Section

348

Acknowledgment

348

References

FIXATIVES

337
337

339

Staining

Acetate Buffer, pH 5.9

328

335

343

SECTION THICKNESS


............................................
!=~~J!t~r. 1.s • ••••••••••••••••••••••••••••••••••••

Cytopreparatory Techniques
352

Cytopreparation

352
352

Collection
GYNECOLOGIC CYTOLOGY

352

NONGYNECOLOGIC CYTOLOGY

353
354
354

Fixation

354
354

Smear Preparation

355

FLUIDS

366

Glossary

372

Index

PRE-FIXATIVES
Saccomanno Fluid

DIRECT SMEARS

355

MUCOID SPECIMENS

356

SPARSELY CELLULAR SPECIMENS

357

FINE NEEDLE ASPIRATIONS

357

SPECIAL PROBLEMS

358

CHOOSING THE BEST METHOD

358

Liquid-Based Cytology

359
360

Cell Blocks

361
361

Cytology Staining

METHODS

HEMATOXYLIN

362

OG-6

362

EA

362
362
363
363
364
364

PAPANICOLAOU STAIN

364

SPECIAL STAINS

364

References

Orange G, 10% Stock Solution
Orange G, Working Solution
TOLUIDINE BLUE WET FILM
Toluidine Blue
CROSS CONTAMINATION

Histotechnology 3rd Edition xv


-

--~

Preface
The reception of the first two editions of this text has far exceeded
my expectations, and I am very grateful that it has found such
a welcome place in the field of histotechnology. The field has
changed, especially in the areas of immunohistochemistry and
instrumentation, since the publication of the second edition, and
there was a need to update the text; therefore I have asked Christa
Hladik, AA, HT(ASCP)cm, QIHC, clinical laboratory manager,
Neuropathology and Immunohistochemistry, UT Southwestern
Clinical Laboratories, University of Texas Southwestern Medical
Center at Dallas, TX, to join me as an author of the third edition.
My experience in these areas has been limited due to my retirement several years ago. All chapters have been carefully reviewed

and most have been updated or expanded. We have attempted to
increase the emphasis on troubleshooting in many areas and have
added numerous illustrations. We are also pleased to add a chapter
on cytopreparatory techniques by Beth Cox, who is certified by
ASCP as both a histotechnician and a specialist in cytology.
It is our hope that this updated edition will continue to serve as
a basic guide for all students of histotechnology, or for practicing
technicians, technologists, residents, and pathologists seeking
to gain a better understanding of the technology utilized in the
histopathology laboratory.

We are especially grateful to Agatha Villegas and Nied
Duckworth for assisting with the preliminary typing of many
chapters; to Charles White III, MD, Director of the Division of

xvi

Neuropathology and Immunohistochemistry and Histology
Laboratories, UT Southwestern Medical Center at Dallas, TX,
for assistance with photographs, chapter review, and mentoring
for the immunohistochemistry and instrumentation chapters; to
Dennis Burns, MD, Division of Neuropathology, UT Southwestern
Medical Center at Dallas, TX, for photomicrographs; to all the
staff at UT Southwestern Medical Center at Dallas, TX, who work
in the Neuropathology, Immunohistochemistry, and Histology
Laboratories and in the gross room at St Paul University Hospital
for their assistance with tissue preparation and staining. Major
contributions were made by the following: Amy Davis, HTL(ASCP),
Debra Maddox, HT(ASCP)QIHC, Ping Shang, HT(ASCP)QIHC,
Pattie Seward, HT(ASCP), Dawn Bogard, HT(ASCP), Courtney

Andrews, HTL(ASCP), Gwen Beasley, HT(ASCP), Eva Osborn,
PA(ASCP), and Chan Foong, PA(ASCP), and Steve Lee, BS,
HT(ASCP).
Our thanks also go to Maureen Doran, HTL(ASCP), Chair of
the Health and Safety Committee of the National Society for
Histotechnology, for reviewing the Safety chapter and offering
many helpful suggestions, and to Robert Lott, HTL(ASCP), who
was able to provide help with images when needed.
Again, to all of you who are students ofhistotechnology, who continue
to search for answers in this field of part art and part science, and
who care first and foremost about the quality of your work on the
specimens entrusted to you, we dedicate this third edition.

.


I
..CHAPTER
.....- . .............................
..................................................... .
~

-·

Fixation

. . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . .. . .

•••••••••••••••••••••••••••••••••

~ -- !l

••••••••••••••••••••

~. ~. ~. ~..~. ! ..•. ~ .~ .~.................................................................... .
On completing this chapter, the student should be able to do the following:
1.

Define the purposes of fixation

2.

Define:
a.
b.
c.
d.
e.

autolysis
fixation
artifact
pigment
nonaqueous fixative
f. coagulating fixative
g. additive fixative - co1v>h 1 ~ •n
h. hypertonic
i. isotonic

3.

4.

Identify the factors that affect the
quality of fixation and describe the
effect of each factor on tissue (eg,
temperature, size of tissue, time of
fixation, or osmolality of fixative)
Identify the properties, functions,
and actions, and determine whether
each action is an advantage or
disadvantage of each of the following
fixative reagents or solutions:
a.
b.
c.
d.
e.
f.
g.

h.
i.

j.
k.

1.
m.
n.

o.
p.
q.
r.
s.
t.

5.

acetic acid
acetone
alcohols
B-5 fixative
Bouin solution
Carnoy and methacarn solutions
formalin (aqueous, buffered,
neutralized, acetate formalin,
formalin alcohol, calcium formalin,
and formalin ammonium bromide)
Gendre solution
glutaraldehyde
glyoxal
Helly solution
Hollande solution
mercuric chloride
Orth solution
osmium tetroxide
paraformaldehyde
potassium dichromate
Zamboni solution

Zenker solution
zinc formalin

d.
e.
f.
g.
h.
i.

Gendre solution
Helly solution
Hollande solution
Orth solution
Zamboni solution
Zenker solution

6.

Identify any special indication for
use of each of the fixatives listed in
objectives 4 and 5

7.

Identify which fixatives require
postfixation washing, and identify
the preferred washing agent

8.

Identify the fixation pigments and
the conditions under which the
pigment may be formed

9.

Identify which of the fixation
pigments can be prevented and
which of the fixation pigments can be
removed

10. For fixation pigments that can be
removed, state the method(s) of
removal; for fixation pigments that
can be prevented, state the method(s)
of prevention
11. Explain the difference between
buffered and neutralized formalin
12. State how paraformaldehyde differs
from formaldehyde
13. Describe the difference between the
terms formalin and formaldehyde
14. Identify the percentage and volume
of formaldehyde in 1,000 mL of a
10% formalin solution

Identify the chemicals in:
a. B-5 fixative
b. Bouin solution

c. Carnoy and methacarn solutions

15. Compare and contrast Zenker and
Helly fixatives

16. List 2 methods of fixation other than
using chemical reagents
17. Identify the preferred method of
fixation (or lack of fixation) for
a. enzyme histochemistry
b. immunofluorescence
c. skeletal muscle cross-striations
(nonimmunohistochemical
staining)
d. pheochromocytomas
e. electron microscopy
f. urates
l· mmunohistochemical methods
tissue for trichrome stains

f'.

18. Identify which fixative reagents are
protein coagulants and which are
noncoagulants
19. Identify which fixative reagents are
additive fixatives and which are
nonadditive
20. If the reagent is an additive
compound, identify the site or group

with which the reagent reacts (if
known)
21. Describe the effect of acetic acid on
erythrocytes and collagen
22. Identify any reagents that have
associated safety hazards and
state the hazard and any special
precautions required
23. Describe the action of zinc in fixation
24. Give the 2 major problems associated
with fixation, and identify at least 3
corrective actions for each

•••• •

Histotechnology 3rd Edition

I


Definition
A fixative alters tissue by stabilizing the protein so that it is resistant to further changes. Baker [1958] uses the following example
to explain fixation: When a door is opened, its position can be
changed easily, but if the door is fixed open, it is altered in such a
way that it is stabilized and is resistant to change. A fixative must
change the soluble contents of the cell into insoluble substances so
that those substances are not lost during the subsequent processing
steps. This change occurs by either chemical (fixative solutions) or
physical (heat, desiccation) means in a process called denaturation.
Denaturation causes the protein molecule to unfold and the internal

bonds to become disrupted. In the process known as additive fixation, this disruption enables the proteir!.JQ__cQmbine chemically
with a fixative molecule. and the protein then hPmmes insoluble
[Feldman 1980] . With nonadditive fixatives (eg, alcohol, acetone), denaturation causes the protein to become less cap::ihle of maintaining
an intimate rel~I

.

I

'

The older definition of fixative action states that a fixative kills,
penetrates, and hardens tissue. Killing will be discussed in the
following section. Penetration is extremely important, because
adequate penetration of the fixative ensures fixation of the interior of the tissue as well as the few exterior cell layers. Hardening
was a very important fixative action in the early days of microtechniques, because much of the sectioning was done freehand.
Because of the array of embedding media available today, other
than to make the tissue firm for grossing, the hardening action is
less important.

Functions of Fixatives
f\

One function of a fixative is to kill the tissue so thatthe-postmortem
activities of decay, or putrefaction (bacterial attack), and autolysis
(enzyme attack) are prevented. Bacterial attack can be prevented in
most fresh tissues by observing very strict antiseptic techniques, but
autolysis cannot be prevented. Autolysis occurs because some of the
enzymes present in tissue continue their metabolic processes, even

after interruption of the blood supply, until something happens
to stop the enzyme action. Some of these metabolic processes
include breaking down cells and therrcomponents. Autolysis is a
very common problem, especially if fixation is delayed in tissues
that are rich in enzymes. Se¥erely autolyzed tissue will fail to stain.
Another function of fixatives is to help maintain the proper
relationship between cells and extracellular substances, such as
the connective tissue fibers (collagen, reticulin, and elastin) and
amorphous ground substance. This stabilization is very important during the subsequent processing steps which might ~er­
wise distort the tissue elements. A fixative also functions to bfing
out differences in refractive indexes and to increase the visibility
of, or the contrast between, different tissue elements. Refractive
index may be defined as the ratio of the velocity of light in air to
the velocity of light in a liquid or solid medium. If air and tissue
had the same index of refraction, the tissue would be invisible;

therefore, enhancing differences in the refractive indexes of
various tissue structures will increase the contrast between those
structures.
Most staining is enhanced by fixation, and frequently tissue that
has not been fixed will stain poorly. Exceptions do exist, as in
the masking of antigenic sites by fixation, thereby decreasing or
completely obscuring antigen sites, resulting in faint or negative
immunohistochemical staining. This effect can be reversed in most
cases by using antigen retrieval techniques. Fixatives also aid in
rerldering cell constituents insoluble, with tissue proteins serving as
the primary target for stabilization. Some fixatives will help stabilize or retain lipids and carbohydrates initially, but much of the time
thes~ ~ubstances will be lost in the subsequent processing. Fixation
will irt'ake the tissue firmer, so that gross dissection and taking
of the thin sections required for processing become much easier.

.

Actions of Fixatives

•l~

• • • • • • • • • • • • • • • • • • • • • •• • \9:,';;,• . .... .

................ .

'
Although very similar to fixative

functions, fixative actions can
be considered a separate topic. Enzymes, which are proteins, are
renderedJD" r tive as a result of the protein-stabilizing action oL
_fixatives. This is a very important fixative characteri~tic.~ecause
enzymatic action causes tissue autolysis. Tissues that are rich
in enzymes, such as liver, pancreas, and brain, are more subject
to rapid autolysis than those tissues with a predominance of
connective tissue fibers. Fixati~es_31so kilLhacteria_and_ molds,
which cause pu~ixatives make tissue mor.e....rec.ey..tiye
to d es aiid,.in._,!I! anµnstances. a t a mordants~"""hich serve
to link the dye to the tissue (mordants are discussM in chapter
6, "Nuclear and Cytoplasmic Staining," pl09). Fixatives modify
tissue con~ioL.ihe maximum retention of form through
subseg\1ent 12rocessing steps€ This is a very important 3ction
because the steps following fixation can induce a dramatic change
in the tissue. The fixative should stabilize the tissue elements so

that the effect of an~~sequ e nt procedures will be minima~k't
Proteins can be2tabilized using various.physical and chemical
methods. One of the physical methocl£.uses h~t. Heat will stabilize
and denature protein, as demonstrated by the cooking of an egg.
Heat fixation generally has not been used in the histopathology
laboratory, but.with.J:he advent gf.the mierowaV€ ~en, it.is finding
much IEQre-use-Microwaves are a form of nonionizing radiation.
When dipolar (charged) molecules, such as water or the polar side
chains of proteins, are exposed to microwaves, the molecules oscillate, or swing back and forth, at the rate of 2.5 billion times per
second. The result is molecular friction or instantaneous heat. The
heat produced is controlled by adjusting the energy levels of the
microwaves and the duration of exposure. Early in the process,
either 1- or 2-stage microwave fixation was used. With 2-stage
fucatim1, the first step involved fixation by immersion in sal_ine_nf
large specime such as the stomach, solid organs, and intestinal
segments to make the tissue sufficiently firm for gross dissection,
and the second step involved the fixation of 2-mm-thick blocks.
immersed in saline and heated to a temperature of 50°C to 68°C
[Leong] or 45°C to SS 0 C: [Hopwood 1993T'IlieSeTeillp~~


critical; if the temperature is allowed to exceed these ranges,-or
-;-maximum of 68°C, the tissue will show pyknotic, overstained
nuclei. Hopwood [1982] stated that the denaturation of proteins that
occurs with overheating can also cause a loss of enzyme activity
and-antigenicity, false localization of nucleic acids, and frequently,
lysis of -red eells. If the temperature used is too low, it will result
in poor fixation . Although saline was widely used for microwave
fixation initially, today the aldehydes, especially formaldehyde, are
more commonly used. Microwave ovens are discussed in chapter

2, "Processing," p39, and chapter 3, "Instrumentation," pp66-68.
[Desiccatio~ is also a physical method of fixing
protein, but is
___,_____
m ciy, if ever, used in routine histopathology. Air-dqci.ng.0£.tm.1~h
pj!~a_ration<

for_Wright staining is probably the most frequent use
of this method of fixation.

The primary ~_[stabilizing protein in the histopathology
laboratory involves the use of 1 or more chemical reagents. These
reagents can be classified as additive or nonadditive and £._oagu~t or noncoag~lant. ~<4!i~a_m:es _fh~~ical_!x~~-9.f,~;.ld
themse ves on, to the tissue~gj,Qn. When a
futive mok'Zt:ile adds ~to a tissue macromolecule, the electrical
charge at the site of attachment may be changed. If the electrical
charge is changed, and that charge was a force helping to maintain
the conformation, or shape, of the protein, then the tertiary structure may be significantly altered. The.J:Q.mmon additive reagents
are mercnrir chlori!k, ~m.trioxi,de,picric acid, formald~
!D:ge. glutaraldehvd~ osmium tf'tro~ide, and zinc.sulfate or~
n
such as aceton~ and the alcohol , ~~-~!:!!s~
raj;._
~lli._~_g_ID.t!Li!. For example, ~and ethyl alcOOol.s
precipitate or coagulate protein but do not add to the tissue. The
primary mechanism by which these fixatives act is tQ)i~~K
_b9uj!d~at UllQ~O~~£S. As a result,
this can cause ~hrink~~nd~~~The amino (-NH) and carboxyl (-COOH) groups on the proteins
are very important in staining. If the fixative adds itself to either

one of these groups, the staining of the tissue will be markedly
affected. At a pH of 7.0, formaldehyde adds on to tissue proteins
primarily at the amino group, with the eventual formation of a
methylene bridge. This results in an excess of negative charges on
the proteins. The heavy metals (chromium, mercury, and osmium)
are cations (positively charged) that combine with anionic (negatively charged) groups of proteins [Sheehan 1980]. This results in an
excess of positive charges. Some of the groups that combine with
cations are sulfhydryl (-SH), carboxyl (-COOH), and phosphoric
acid (-POJ This will be discussed further under each fixative
reagent.
To better understand the coagulant and noncoagulant action of
fixatives, imagine 2 dishes, with 1 dish containing a piece of gelatin
(eg, Jello) and the other dish containing a mesh ball. Which of the
substances in the dishes do you think aqueous or alcoholic solutions would penetrate or enter most freely? An aqueous solution
would easily enter all of the crevices in the mesh, but would have
a difficult time entering, or penetrating, the gelatin. Coagulation
establishes a network in tissue that allows solutions to readily penetrate or gain entry into the interior of the tissue. The noncoagulant

fixatives act by creating a gel that makes penetration by the subsequent solutions difficult. Because the noncoagulant fixatives do
not allow good penetration by the reagents applied after fixation
(during processing), Baker [1958] considered these fixatives inferior
for paraffin infiltration and embedding. Although the importance
of this phenomenon is really seen at the microscopic level, it can
be demonstrated at the macroscopic level. Wenk demonstrates this
phenomenon with students as follows: take small jars with lids
(50-mL beakers will also work) and put 20 mL of a different fixative
in each jar; label carefully. Use whatever fixatives are readily available in the laboratory, but be sure to include 10% formalin, aqueous
zinc formalin, acetone, alcohol, and acetic acid. Separate a raw
egg at room temperature, saving only the white, which is protein;
pipette 2 mL of the egg white into each fixative solution. See what

happens by watching the change in consistency of the egg white,
and the time frame for any changes to occur [ii.I], [il.2], [il.3].
The coagulant fixatives are zinc salts, mercuric chloride, cupric
sulfate, ethyl alcohol, methyl alcohol, acetone, and picric acid.
Baker [1958] classified acetic acid as a coagulant of nucleic acids,
but a noncoagulant of cell cytoplasm; however, Wenk [2006] found
that acetic acid acted as a coagulant of egg white. The noncoagulant fixative reagents are formaldehyde, glutaraldehyde, glyoxal,
osmium tetroxide, and potassium dichromate. For use after a
noncoagulant fixative, infiltration or embedding media other than
paraffin (eg, plastics) work best.
A summary of fixatives categorized by composition and properties
is shown in [fl.I, p 4].
Knowledge of fixatives and fixation has evolved over time, beginning with the biologic effects of mercury and its salts dating back
to Hippocrates [Bancroft 1982]. Wine, or alcohol, also has long been
recognized as a preservative. Many fixatives that differed only
slightly were developed in the 19th century. Gray [1954] listed more
than 500 fixatives, with only a few of these being widely accepted.
Baker [1958] introduced the convention of naming the fixative solution after its first user and disregarding any minor modifications.

[i I . I] The egg white hardens and turns white almost immediately in the
I00% alcohol and in the acetone, similar to raw egg on a hot skillet. The
photograph was taken I0 minutes after the egg white was placed in these
two solutions, but the change was seen within I minute. Within 2 hours, the
egg white was so hard it was brittle and would break apart when touched
with a wooden stick. (Reprinted with permission from Wenk [20061)

Histotechnology 3rd Edition 3


FIXATIVES

Additive

~

Non -additive

Alcohols
Acetone
Ace tic acid
/

Noncoagulants
For maldehyde
Glutaraldehyde
Glyoxal
Osmium tetroxide/
osmic aci d
Potass ium dich roma te

[i 1.2] In the Bouin solution and the zinc formalin, the egg white hardens a
little slower than it does in pure ethanol or acetone, but the egg white has
a consistency of a soft-boiled egg after I 0 minutes of fixation . There is less
hardening in these fixatives than in the pure ethanol or acetone. Hollande
solution, mercuric fixatives, and I00% acetic acid behave similarly. (Reprinted
with permission from Wenk [20061}

\

Coagulants

Mercu ric chloride
Chromic acid
Picric acid
Zinc salts
Cupric salts

I

Alcohols
Acetone
Acetic acid (texts differ)

[fl.I] A summary of the additive vs nonadditive fixatives , and coagulant vs
noncoagulant fixatives .

for electron microscopy; however, some laboratories have moved
away from the use of cold fixation. Some parts of the cell are
less affected when formaldehyde fixation is performed at room
temperature instead of refrigerator temperature, and we prefer
the ultrastructural preservation yielded by room temperature
fixation [Carson 1972]. Today higher temperatures are being used
for fixation in both tissue processors and microwave ovens; in
general, increasing the temperature of the fixative up to about
45°C is reported to have very little effect on tissue morphology.

SIZE

[i 1.3] The egg white is at the bottom end of the wooden sticks but
cannot be seen. The egg whites have not changed color or hardened in
the first 10 minutes. The egg white in the 10% formalin looks and acts as

ii it has been put into room-temperature water; it continues to have the
same consistency as raw egg white. Even by the next morning it is not
visible, has not hardened, and is not dissolved; it remained clear and could
be swirled. Egg white in glutaraldehyde behaves similarly. In the alcoholic
formalin, the egg white remains the same for the first few hours, but
eventually the 70% alcohol causes the egg to slightly turn white in some
areas and harden slightly; however, the alcoholic formalin never hardens
the egg completely, no matter how long it is in this fixative. (Reprinted

The thickness of the tissue is especially important because of its
effect on reagent penetration. Size should be considered when
the gross tissue specimens are placed in fixative. If large specimens such as segments of colon or small intestine are held for
any extended period without being surgically opened to expose
all layers, the fixative will have difficulty penetrating through the
entire wall to the inner epithelial surface. The result frequently
is autolysis of the epithelium; therefore, specimens of this type
should be opened before they are placed in fixative solution. A
more common consideration is the size of the sections cut for
processing. For routine processing schedules, sections should be no
more than 3 mm thick. When processing on a short protocol, the
sections must be even thinner or the reagents will not completely
penetrate the section. At no time should a section be so thick that it
touches both the top and bottom of the tissue-processing cassette.

with permission from Wenk [2006])

VOLUME RATIO

Factors Affecting Fixation
............................................

Fixation factors are those elements that affect the quality of fixation; most of them are easily controlled.

TEMPERATURE

The temperature at which fixation is carried out may affect tissue
morphology. In general, an increase in temperature increases
the rate of fixation but also increases the rate of autolysis and
diffusion of cellular elements. Traditionally, 0°C to 4°C has been
considered the ideal temperature for the fixation of specimens
4 Fixation I Ch I

The ratio of the tissue volume to the fixative volume is one of the
fixation criteria over which there may be limited control. The fixative volume should be at least 15 to 20 times greater than the tissue
volume. Effects of many fixatives are additive; fixative molecules
are bound chemically to the tissue, and the solution is gradually
depleted of these molecules. Tissue also contains soluble salts that
are dissolved by the fixative solution. The "2-way exchange" does
not greatly alter the characteristics of the fixative if a large volume
ratio is used [fl.2a]; however, if the volume of the tissue is greater
than that of the solution [fil.2b], the fixative composition can be
altered; therefore, the volume ratio is a very important consideration. Frequently, staining problems are really the result of poor
fixation because of the use of an inadequate volume of fixative.


b

a

ff
f ff ff f

f .
fff
ffffffff f"
- - tissue - - f f f f f ff ff
-

fixative ·-

-

[fl.2] Soluble salts "s" are dissolved out of the tissue into solution in
the fixative, whose molecules "f" attach to the tissue, decreasing fixative
concentration. This is oflittle consequence if the fixative-to -tissue ratio is large a,
but the fixative's composition can be markedly altered if the ratio is small b.
[fl.3]

A section of small intestine.

TIME

Time is important in 2 respects. The first consideration is the
interval between interruption of the blood supply and placement of the tissue in fixative. Ideally, the tissue should be placed
in fixative immediately after surgical removal, and autopsies
should be performed immediately after death. The more time that
elapses between interruption of the blood supply and fixation,
the more postmortem changes that can be demonstrated microscopically. [fl.3] and [il.4] illustrate well-preserved tissue, and
[il.5] illustrates postmortem changes in the same type of tissue.
The cellular detail that can be seen in a well-preserved section of
small intestine is illustrated in [fl.3]. The outer, relatively monotonous layer of cells is the epithelium, which is defined as a membrane
that covers or lines. Notice that the section shows 2 fingerlike

projections of the small intestine. These fingerlike projections
are called villi, and they are a very distinctive feature of the small
intestine. When you see them, you can confidently identify the
tissue as small intestine. Intestinal epithelium is composed of a row
of simple columnar cells and an occasional goblet cell, 1 of which is
identified. Between the fingerlike projections are crypts with cells
containing an abundance of secretory granules that usually stain
a deep red with eosin; these are Paneth cells. The tissue underlying
the epithelium is called the lamina propria. It contains connective
tissue cells and fibers, very small blood vessels, and nerve twigs.
Although none is illustrated, an aggregate oflymphocytes called a
lymph nodule will be present occasionally. Underlying the lamina
propria is a layer of smooth muscle, the muscularis mucosa. The
epithelium, the lamina propria, and the muscularis mucosa form
the mucosa, the first of 4 layers common to the gastrointestinal
tract. Because autolytic changes and bacterial decomposition are
most pronounced on the mucosa, only this layer is described.
A section of small intestine in which all of the structures identified thus far are well preserved is shown in [il.4]. This section was
taken from a surgical specimen that was opened and placed in
fixative solution immediately after removal. Therefore, the fixative
had early contact with the epithelium and was able to penetrate
from both the epithelial and the serosal (outermost) surfaces. This
rapidly halted the postmortem changes of autolysis and putrefaction. Notice the well-preserved epithelium and compare this illustration with [il.5] in which the epithelium is entirely gone except
in a few deep glands. The lamina propria is completely denuded, a

[i 1.4] The mucosa is excellently preserved in this section of small intestine.
Note that autolysis is absent and the epithelium is intact.

[i 1.5] Fixation of this section of small intestine taken at autopsy was delayed.
Marked autolysis has occurred, and except for a few glands, or in the crypts,

the epithelium is gone. Most of the goblet cells and the argentaffin cells have
disappeared. Only the denuded lamina propria of the villi can be seen.

common finding in autopsy sections of the gastrointestinal tract.
This section is autolyzed; autolysis will cause desquamation of the
epithelium and separation from the basement membrane [Leong
1994]. Because of the bacterial content of the gastrointestinal tract,
some of the changes seen in this section are probably the result
of putrefaction. When selecting control tissue, one must be very
Histotechnology 3rd Edition 5


careful about using autopsy tissue. For example, the tissue shown
in [il.5] would not be a good control for mucin stains, because
the mucin-containing goblet cells have not been preserved.
The duration of fixation is also important. The current trend of
decreasing the time allowed for fixation is resulting in many problems. Adequate fixation is needed so that the tissue will not be
distorted by the subsequent processing steps. [il.6] shows a tissue
specimen that is difficult to identify. It is a section oflung that was
not well fixed, and proper relationships of tissue structures have
not been maintained during the subsequent processing steps. [il.7]
shows a well-fixed section oflung. Tissue that is not well fixed does
not process well, and subsequently will not stain well, so adequate
fixation time is of primary importance in quality assurance. [il.8]
also contrasts poorly fixed, undifferentiated tumor tissue with a
well-fixed tissue section from the same tumor [il.9]. The latter
section shows nuclear bubbling that is commonly attributed to
fixation with formalin alone [Banks 1985]; however, Dapson [1993]
attributes it to the specimen not being completely fixed before dehydration is begun. Formalin should have at least 6 to 8 hours to act
before the remainder of the processing schedule is begun. Dapson

[2004] reported that in a carefully controlled study in his laboratory,
artifact-free sections could be produced only after a minimum of
30 to 40 hours of fixation with neutral-buffered formalin, and
marked artifacts were present after only 7 hours formalin exposure. Much of the processing occurring today takes place in the
dehydrating alcohols, because not enough time is allowed for fixation to occur in the fixative solution. Dapson also stated that with
proper fixation, the tissue is almost immune to artifacts; whereas,
with incomplete fixation, the specimen is vulnerable to the effects
of any subsequent denaturing agent, be it chemical or physical.
The importance of time in fixation was stressed, when in 2007,
the American Society of Clinical Oncology and the College of
American Pathologists released guidelines to improve the accuracy
of testing for human epidermal growth factor receptor 2 (HER2)
in invasive breast cancer [Wolff 2007]. The guidelines recommend
that the incisional and excisional biopsy specimens used for HER2
testing be fixed in 10% neutral-buffered formalin for a minimum
of 6 hours and a maximum of 48 hours, stating that prolonged
fixation may show false-negative results.

[i 1.6] A section of lung that was not completely fixed before processing
shows poor stabilization of the tissue structures, and proper relationships are
not maintained.
6

Fixation I Ch I

[i I. 7] A section of lung that was well-fixed before processing shows the
proper relationships of tissue structures.The interalveolar septa are well
preserved and the alveolar sacs are clearly seen.A bronchiole with wellpreserved epithelium is seen in the upper left corner.

[i 1.8] A section of an undifferentiated tumor that has not been well

fixed shows that the proper relationship of cellular elements has not been
maintained.The staining is poor, with a lack of contrast between the cell
nucleus and cytoplasm.

[i I. 9] A section from the same tumor as seen in [i 1.8] that has been well
fixed in I0% neutral-buffered formalin. The nuclei show the "bubbling artifact"
frequently associated with formalin fixation. Note that the contrast between
the cell nucleus and cytoplasm is much better, and crisp nuclear membranes
are demonstrated.


While tissue must be left in most fixatives for an adequate length
of time to achieve good fixation, tissue cannot remain indefinitely
in many fixatives. Tissue must be removed from fixatives such
as glutaraldehyde, Helly solution, Zenker solution, and Bouin
solution; washed if indicated; and then stored in an appropriate
storage solution. If allowed to remain in these fixatives too long,
the tissue becomes overhardened and staining may be impaired.

CHOICE OF FIXATIVE

The broad range of fixative choices requires the technician to stop
and think, on receipt of the specimen in the laboratory, about
which fixative is appropriate. If tissue is improperly fixed for a given
technique, frequently no corrective action is possible. Therefore,
immediately upon presentation of the specimen, the method
of fixation must be chosen. Sometimes no fixation is desired; if
an immunofluorescence study or an enzyme profile is needed,
the specimen must be frozen without fixation. Although some
enzymes can be demonstrated on frozen sections that have been

fixed, other enzymes are rapidly inactivated by even brief contact
with a fixative . Some antibodies used in immunohistochemical
procedures require that tissue be frozen, sectioned, and then
left unfixed or briefly fixed in acetone. A more comprehensive
discussion of tissue fixation for immunohistochemical studies is
found in chapter 12, "Immunohistochemistry," p279.
Often, a particular fixative must be chosen to ensure optimal
demonstration of a particular tissue element, such as the choice
of Zenker solution when muscle cross-striations are to be stained
with phosphotungstic acid-hematoxylin (PTAH) or Bouin solution when the tissues are to be stained with a trichrome technique.
To increase the staining reaction, a microscopic section of tissue
that has been fixed with 1 reagent frequently can be treated with
another fixative reagent. This process is called postfixation or
mordanting, and is used in the Masson trichrome technique, in
which a microscopic section of formalin-fixed tissue is mordanted
with Bouin solution before staining. Although postfixation gives
very good results with the Masson technique, superior staining
can be achieved with some techniques only when the tissue is
fixed appropriately at the outset. Some tissue elements cannot be
demonstrated if the original fixation is incorrect. For example, the
demonstration of chromaffin granules, found in cells of the adrenal
gland, is helpful in the identification of pheochromocytomas, but
these granules cannot be demonstrated after formalin fixation.
For the subsequent demonstration of chromaffin granules, tissue
must be fixed in a primary dichromate fixative such as Orth solution. Urate crystals are water-soluble and require a nonaqueous
fixative such as absolute alcohol. The proper fixative also must be
used if electron microscopy or ultrastructural studies are required.

PENETRATION

Fixative solutions penetrate at vastly different rates. According to
Baker [1958], the factors that determine the minimum length of
time that a fixative should act are the rate of penetration and the
mode of action. Most coagulant fixatives achieve their full effect
on tissue at any particular depth as soon as they have penetrated
to that depth at a concentration sufficient to cause coagulation.
Formaldehyde, a noncoagulant fixative, penetrates fast, but

continues to cross-link proteins for a long time after the penetration is complete. In fact, according to Baker [1958], formaldehyde
penetrates faster than any of the common fixative ingredients.
Fixatives in order of decreasing speed of penetration are as follows:
formaldehyde, acetic acid, mercuric chloride, methyl alcohol,
osmium tetroxide, and picric acid. Although the information is
not available, ethyl alcohol probably penetrates at a rate similar
to methyl alcohol. The rate of penetration is affected by heat, but
not by the concentration of the fixative. Because fixation begins
at the periphery of the tissue and proceeds inward, most of the
interior fixation oflarger specimens may be due primarily to only
1 chemical in a compound fixative.

TISSUE STORAGE

The method of wet tissue storage is very. important because the wet
tissue often will be needed for additional studies. If the tissue has not
been fixed and stored properly, additional studies may be impossible.
Storage is not usually a problem with tissue fixed in neutral-buffered
formalin because the tissue may remain in this solution indefinitely; this is not true of many other fixatives. However, if immunohistochemical stains are anticipated at a future time, tissue should
be transferred from formalin to 70% alcohol to stop cross-linking.
Appropriate storage is described in the individual sections on each of
the more common fixative solutions.

pH
The pH of the fixative is not very important in light microscopy
and many fixatives are quite acidic. Varying the pH from 4 to 9
apparently makes little difference in the fine structure produced
by formalin fixation; however, a pigment is produced at a lower
pH. The pH of the fixative solution is very important in electron microscopy. When ultrastructural preservation is the main
purpose of fixation, the solution should be buffered to a pH of 7.2
to 7.4. This is a physiological pH, that is, approximately the pH of
tissue fluid.

OSMOLALITY

Osmolality refers to the number of particles in solution and is not
as important in light microscopic studies as in ultrastructural
studies. Body fluids have an osmolality of about 340 mOsm or
0.3 Osm. A 1-0sm solution may be defined as 1 formula weight
of a nondissociating compound (eg, sucrose) per 1,000 g of solution. 1 formula weight of a dissociating compound (eg, sodium
chloride) per 1,000 g of solution is equal to a 2-0sm or 2,000mOsm solution. The terms isotonic, hypotonic, and hypertonic
are used frequently; normal (isotonic, physiological) saline solution is sometimes used in histopathology as a holding solution
for tissue. What does this mean and why is it important? [fl.4a]
shows a cell in a solution that is more concentrated or contains
more particles than the cell cytosol; this solution is hypertonic
to the cell. The cell membrane (plasma membrane) is a semipermeable membrane that allows water molecules to pass through it
very readily. Water passes through the cell membrane toward the
most concentrated solution in an effort to equalize the concentrations on both sides of the membrane. When surrounded
by a hypertonic solution, the water leaves the cell and the cell
Histotechnology 3rd Edition

7

a

b

Reactions of the Cell with Fixatives

THE NUCLEUS

[fl.4] The effect ofhypertonic solution on cells. a A cell in a hypertonic
solution; b The cell showing shrinkage because water was drawn from the cell
into the surrounding solution.

a

b

[fl.5] The effect ofhypotonic solution on cells. a A cell in a hypotonic
solution; b The cell showing swelling because water was drawn from the
surrounding solution into the cell.
shrinks [fl.4b]. If the cell is placed in a hypotonic solution or
one that contains fewer dissolved particles than the cell cytosol
[fl.Sa], the cell swells, possible rupturing its membrane (fl.Sb].

Deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and
attached protein are found in the nucleus. Much more is known
about the effect of fixatives on proteins than on nucleic acids.
Although several fixatives are used for nucleic acids, most fixatives do not appear to react chemically with them. Acetic alcohol
and Carnoy solution are the preferred fixatives for nucleic acids;

formaldehyde does not react with DNA and RNA in their native
states until the temperature reaches about 45°C for RNA and
65°C for DNA [Hopwood 1993]. Much of nuclear fixation is probably
entrapment of RNA and DNA molecules by the fixed or stabilized
nuclear proteins. Banks states that the coagulating or precipitating
fixatives render tissue more resilient to the disruptive effects of
sectioning, deparaffinization, and staining. This results in shaper,
more intact-appearing nuclei. Following formalin fixation, the
nuclei often show coalescence of the chromatin into strands with
intervening clear spaces. This has been called nuclear bubbling
[il.9, p6]; Banks states that nuclear bubbling is introduced in the
deparaffinization step on formalin-fixed tissue, because the nuclei
are only delicately fixed.

PROTEINS

Often it is the osmolality of the fixative vehicle, or the solution
exclusive of the fixative ingredient, that is critical. Water is the most
rapidly penetrating component of an aqueous fixative, so the central
parts of a specimen are probably in contact with a hypotonic solution
before fixation occurs. Unreactive salts with small rapidly diffusing
ions (eg, sodium sulfate or sodium chloride) frequently are added to
fixative mixtures to prevent the damage caused by these hypotonic
solutions [Kiernan 1999]. Formaldehyde is not osmotically active, so
although 10% neutral-buffered formaldehyde solutions appear to be
very hypertonic (approximately 1,800 mOsm), most of the tonicity is
related to the osmotically inactive formaldehyde molecules.
As mentioned before, physiological saline solution can be used as
a holding solution, and other isotonic solutions with a salt composition more closely approximating that found in body fluid also
may be used. However, even though they are isotonic, these solutions are not without effect on the tissue and should not be used

for prolonged holding of tissue. For biopsy specimens that cannot
be placed in fixative immediately, it is probably a better practice
to dampen a piece of gauze with saline solution, squeeze out the
excess, and place the tissue on the dampened gauze. Tissue treated
in this way can be sealed in a plastic container and placed on ice
for short-term holding. Kidney biopsy specimens for immunofluorescence frequently are held, or even mailed, in Michel transport
solution. The formula and directions for use are given on p23,.

Most nonnuclear staining occurs because of the proteins present
and the particular chemical group or groups with which a fixative reacts. Proteins have a primary, secondary, and tertiary structure. The primary structure is determined by the arrangement
of covalent bonds in the amino acid sequence. The secondary
structure is determined by hydrogen bonding between various
components of the peptide chain, and the tertiary structure is
defined as the total 3-dimensional structure. Hydrogen bonds,
ionic (electrostatic) bonds, hydrophobic bonds, and disulfide
bonds are responsible for the tertiary structure of a protein
[Pearse 1980]; these folded conformations are generally very
fragile. Additive fixatives can alter the 3-dimensional shape of
proteins by changing electrical charges at the site of attachment.
The nonadditive, coagulant fixatives cause proteins to become
insoluble by altering their tertiary structure. Pearse [1980] states
that methanol and ethanol preserve the secondary structure of
proteins while markedly affecting their tertiary structure. The
isoelectric point of the proteins may be shifted by the reaction.
If they are known, the sites of fixative attachment will be pointed
out as each fixative is described. The effects of the attachment on
hematoxylin and eosin (H&E) staining are also be discussed in
chapter 6, "Nuclear and Cytoplasmic Staining," pll4.

LIPIDS

The factors that influence fixation are very important in quality
assurance because improper fixation cannot be corrected in subsequent processing steps; instead, these subsequent steps further
differentiate the products of fixation.

8 Fixation I Ch I

While several of the fixatives will preserve lipids, only 2 chemicals
will fix lipids so that they are not lost in the subsequent processing
steps. These are osmium tetroxide and chromic acid. The chemical
reactivity of lipids is altered by both of these reagents.